(a) Field of the Invention
This invention relates to detection of Helicobacter pylori (hereinafter called H. pylori), source bacterium of gastrointestinal disorders, and in particular relates to an H. pylori detecting composition produced by applying a phenate method for ammonium ion's quantitative determination to a conventional urease enzyme test and relates to a detecting kit and method using this composition.
(b) Description of the Related Art
H. pylori is noted as a source bacterium causing gastrointestinal disorders including gastric cancer and gastroduodenal ulcers, and the WHO has prescribed H. pylori as a typed carcinogen. Precise diagnosis, i.e., accurate detection of H. pylori, should be promoted to cure gastrointestinal disorders caused by this H. pylori. H. pylori detecting methods being used up to now can be divided mainly into two types, i.e., non-invasive tests not using an endoscope and invasive tests using an endoscope to get biopsy tissue for detection purposes.
Non-invasive tests include serology tests and .sup.13 C or .sup.14 C Breath tests. Serology tests measuring H. pylori antibodies have a problem in that the existence of the antibody does not necessarily mean that diseases are developing because it takes more than 6 months for antibody levels to drop after the cure of diseases. The .sup.13 C or .sup.14 C Breath test uses the principle that if urea marked with .sup.13 C or .sup.14 C is ingested, it is transformed into marked CO.sub.2 in the stomach if H. pylori exists and detected in exhaled air. However, the method is not usually used because it involves high cost equipment.
On the other hand, invasive tests are more commonly used to detect H. pylori. Invasive tests detecting methods using biopsy tissues samples collected during an endoscoping includes histology, the PCR (Polymerase Chain Reaction) method, culturing, and the urease enzyme test. Histology is a method confirming the existence of H. pylori through general tissue research, the PCR method is a method confirming the existence of H. pylori by using chain reaction of polymerization enzyme, and culturing is a method of directly cultivating H. pylori from biopsy tissue. However, there is a problem in that all these methods are difficult and take much time, so they can not be used commercially.
The urease enzyme test, which can be used easily in the endoscope chamber and is the most efficient method, uses the fact that H. pylori produces urease enzyme which has a much higher degree of activity compared to other microorganisms. That is, if urease exists urea added in the detecting kit decomposes and ammonia is produced to cause pH to be increased making a pH indicator react and change color. A H. pylori detecting method measures the activity degree of urease like this. Commercialized kits using the urease enzyme test now are "CLOtest" (U.S. Pat. No. 4,748,113), "Hpfast" (U.S. Pat. No. 5,439,801) and "Pyloritek" (U.S. Pat. Nos. 5,314,804 and 5,420,016). Compositions used in "CLOtest" include 10.about.40 g/l of urea, 1.about.5 g/l of bactericide, an available quantity of pK.sub.a 6.5.about.8.5 indicator (phenol red) and the remainder being water, having a 5.0.about.6.5 pH. However, the CLOtest has problems in that the positive rate vary with the readers because the reaction speed of urease that the above compositions and H. pylori produce is slow. Therefore, diagnosis is possible only after about 24 hours and it is not possible to obtain diagnostic results on the endoscope examination date which requires that the patient inconveniently visit the hospital once more. Furthermore, the various colors appearing after more than 24 hours makes accurate diagnosis difficult. Hpfast is fundamentally similar to the CLOtest, but different in that cell wall detergent is added and uses as an indicator mixture of phenol red, methyl red, and bromothymol blue. With Hpfast, an H. pylori positive is determined from a dark green color of pH 6.2 and a light green color of pH 6.0 is determined as an H. pylori negative. However, the accurate reading of this dark green and light green colors is difficult. Pyloritek uses a multilayer test device differently from the above mentioned CLOtest and Hpfast, the main characteristics of this is that the urease reacting part and reacted product as an ammonia detecting part are on different layers and the pHs of each layers are different. Although Pyloritek can produce diagnosis results within one hour, the false positive rate increases if the determination time exceeds one hour. Therefore, there is an increasing possibility of false positives if an accurate reaction time is not adhered to during the endoscope examination.
Moreover, the above mentioned urease enzyme methods including the CLOtest, Pyloritek, etc. could also react with the small amounts of low activity urease which bacilli such as Staphylococcus hominis, Streptococcus salivarius, E. aerofaciens, L. fermentum, etc. have and false positive rates could be increased in cases of long decision times.